JP2020524689A - Plasminogen treatment of conditions associated with PAI-1 overexpression - Google Patents

Abstract

The present invention relates to the use of plasminogen, variants thereof, or analogs thereof having plasminogen activity, for the prevention or treatment of conditions or diseases characterized by increased PAI-1 levels. Conditions and diseases characterized by increased PAI-1 levels are re-categorized into two categories: diseases associated with diminished remodeling capacity of blood vessels or tissues and metabolic and hormonal disorders associated with increased PAI-1 levels. being classified.

Description

The present invention relates to the field of medicine. Certain aspects of the invention use, prevent, delay progression, and related methods of treatment of plasminogen for preventing, slowing, and treating conditions in which PAI-1 levels are compromised or therapeutic indications. , And the use of plasminogen as a PAI-1 modulator.

It is known that some conditions and diseases are characterized by PAI-1 overexpression in injured tissue or PAI-1 overexpression in blood. Drugs known to lower plasma levels of PAI-1 include angiotensin converting enzyme (ACE) inhibitors, insulin sensitizers, and hormones used in female hormone replacement therapy.

In a tissue in which PAI-1 is overproduced, a significant influence on the remodeling ability of blood vessels and tissue is observed (Carmeliet et al. 1997, Inhibitory role of plasma inhibitor inhibrator-1 in arterial wound healing inflation). targeting and gene transfer study in mice. Circulation 96:3180-3191). Progressive accumulation of extracellular matrix (ECM) in the glomerulus and stroma is almost universally seen in chronic renal disease, regardless of underlying disease. There is considerable evidence that reduced ECM degradation contributes to matrix accumulation and that ECM degradation is primarily controlled by plasminogen activators/systems. Plasmin is directly by degrading matrix proteins such as fibronectin, laminin, proteoglycans, and type IV collagen, and fibrin, and by converting an inactive matrix metalloproteinase into an active form that degrades collagen proteins. , Indirectly contributes to the decomposition of ECM. The most widely studied diseases that are believed to be affected by PAI-1 include glomerulosclerosis and tubular interstitial fibrosis (Eddy 2002, Plasminogen activator inhibitor-1 and the kidney. Am. J. Physiol. Renal Physiol. 283: F209-220), Lung fibrosis (Olman et al. 1995, Changes in procoagulant and fibrinogenic essence incl. -1630), glomerulonephritis (Keeton et al. 1995, Expression of type 1 plasmaminactivator inhibitor in renal tissue squirt in a moisturine ind. mice with autoimmune disease acquire a hypofibrinolytic / procoagulant state that correlates with the development of glomerulonephritis and tissue microthrombosis.Am.J.Pathol 151 (3):. 725-734), atherosclerosis (Schneiderman et al.1992, Increased type 1 plasminogen activator inhibitor gene expression in atherosclerotic human arteries. Proc. Natl. Acad. Sat. cit. USA. -1 expression in human liver and health or atherosclerotic vessel walls. Thromb Haemost. 72(1):44-53), Chronic Obstructive Pulmonary Disease (COPD) (Wang et al. 2016, Elevated circulatory erosion regulation, swelling, circulation, and swelling). Int. J. Chron. Obstruct. Pulmon. Dis. 11: 2369-2376), chronic kidney disease (CKD) (Eddy and Fogo 2006, Plasminogen activator inj. Am. Soc. Nephrol. 17(11):2999-3012), membranous nephropathy (Edy and Fogo 2006), and chronic allograft nephropathy (Edy and Fogo 2006).

Studies have confirmed that the extent of fibrosis corresponds to the pattern and extent of PAI-1 expression (Oikawa et al. 1997, Modulation of plasmaminogen activator-1 in vivo: a new mechanism for-the-fore. fibrotic effect of renin-angiotensin inhibition. Kidney Int. 51: 164-172). Therefore, evidence is that affecting PAI-1 expression affects fibrosis, including arteriosclerosis, nephrosclerosis, pulmonary fibrosis, myelofibrosis (Vauguhan 2011) and other fibrosis. Overexpression of PAI-1 is observed in the lungs of patients with idiopathic pulmonary fibrosis and is mainly concentrated in injured or inflamed tissue.

Fibrosis is defined as a disease associated with fibroproliferative or abnormal fibroblast activation. Deregulation of wound healing leads to excessive activation of fibroblasts and excessive accumulation of extracellular matrix (ECM) proteins in the wound area, pathological manifestation of fibrosis. Accumulation of excessive levels of collagen in the ECM depends on two factors: increased collagen synthesis rate and/or decreased collagen degradation rate due to cellular proteolytic activity. Urokinase/tissue-type plasminogen activator (uPA/tPA) and plasmin play important roles in cellular proteolysis of ECM proteins and maintenance of tissue homeostasis. The activities of uPA/tPA/plasmin and plasmin-dependent MMPs are largely dependent on the activity of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of uPA/tPA. Under normal physiological conditions, PAI-1 regulates the activity of uPA/tPA/plasmin/MMP proteolysis, thereby maintaining tissue homeostasis. Elevated levels of PAI-1 during wound healing help promote wound healing by inhibiting uPA/tPA/plasmin and plasmin-dependent MMP activity. In contrast to this scenario, under pathological conditions, excess PAI-1 contributes to the excessive accumulation of collagen and other ECM proteins in the wound area, thus preserving scars. Although levels of PAI-1 are significantly elevated in fibrotic tissue, the lack of PAI-1 protects various organs from fibrosis in response to injury-associated pro-fibrotic signals. Therefore, PAI-1 has been implicated in the pathology of fibrosis in various organs including heart, lung, kidney, liver, and skin (Ghosh and Vaughan, 2012).

In addition to the above conditions, disorders of PAI-1 levels are also associated with metabolic/hormonal disorders such as obesity, polycystic ovarian disease; and other disorders such as amyloidosis (Vaughan 2011), Alzheimer's disease (Oh et al. 2014). , Plasminogen activator inhibitor-1 as an early potential diagnostic marker for Alzheimer's disease. Exp. Geronol. Perhaps the most common clinical condition associated with increased plasma PAI-1 production is obesity. Analytical studies of the upstream regulatory region of the PAI-1 gene include a glucocorticoid response element (GRE) that also localizes aldosterone responsiveness (4), a very low density lipoprotein (VLDL) response site, related transcription-responsive site (Eriksson et al.1998, Very-low-densisty lipoprotein response element in the propomter region of the human plasminogen activator inhibitor-1 gene implicated in the impaired fibrinolysis of hypertriglyceridemia. Arterioscler. Tromb.Vasc. Biol.18 : 20-26), and two Sp1 sites that are believed to mediate glucose-glucosamine responsiveness (Chen et al. 1998; Chem. 273: 8225-8231).

There is a need in the art to discover PAI-1 modulators and their use for the treatment of numerous therapeutic indications associated with abnormal PAI-1 levels.

Although reporting several diseases that may be associated with increased levels of PAI-1, Vaughan 2011 directs future research towards the development of synthetic orally active PAI-1 antagonists. Nevertheless, Vaughan 2011 warns against complete inhibition of PAI-1, which indicates that PAI-1 causes intracranial and joint bleeding after mild trauma, delayed surgical bleeding, severe bleeding. This is because it may cause menstrual bleeding and abnormal bleeding such as frequent bruising. Therefore, there is a need for compounds that do not completely inhibit or antagonize PAI-1 but reduce PA-1 expression to normal levels.

The inventors have discovered PAI-1 modulators that down-regulate PAI-1 overexpression without completely inhibiting PAI-1. The PAI-1 modulators we have found are completely natural rather than synthetic. It is plasminogen. Surprisingly, we found that administration of exogenous plasminogen to a subject with normal endogenous plasma plasminogen activity levels resulted in PAI-1 expression in injured tissue in which PAI-1 was overexpressed. Was found to be successfully down-regulated. Administration of exogenous plasminogen may suppress overexpression of PAI-1 in the lungs of subjects with idiopathic pulmonary fibrosis (IPF), allowing extracellular matrix (ECM) degradation. know.

A general aspect of the invention is the use of plasminogen, a variant thereof, or an analog having plasminogen activity for the prevention, delay of progression or treatment of a condition or disease characterized by increased PAI-1 levels. Regarding pharmaceutical use. A general aspect of the invention is also the prevention, delay of progression of conditions or diseases characterized by elevated levels of PAI-1 comprising administration of plasminogen, variants thereof, or analogs having plasminogen activity. Alternatively, it relates to a method of treatment. A general aspect of the invention is further plasminogen having plasminogen activity for the preparation of a medicament for the prevention, delay of progression or treatment of a condition or disease characterized by increased PAI-1 levels, It relates to the use of the variant, or an analogue thereof. Conditions and diseases characterized by increased PAI-1 levels include, but are not limited to, diseases associated with decreased remodeling capacity of blood vessels or tissues, and metabolic disorders and hormones associated with increased PAI-1 levels. It is reclassified into two categories: disability.

Diseases associated with a reduced remodeling capacity of blood vessels or tissues that may be prevented or treated by the present invention or that slow its progression include, but are not limited to, chronic renal disease, glomerulosclerosis, fibrosis. , Tubular interstitial fibrosis, pulmonary fibrosis, glomerulonephritis, atherosclerosis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, idiopathic pulmonary fibrosis, chronic kidney disease (CKD), membrane Include nephropathy, or chronic allograft nephropathy.

Metabolic or hormonal disorders associated with the ability to prevent or treat increased PAI-1 levels or delay its progression according to the present invention include, but are not limited to, obesity, polycystic ovarian disease, amyloidosis. , Alzheimer's disease, alopecia, and aging.

The present invention relates to the following items.
1. Use of plasminogen, a variant or analogue thereof, for the preparation of a medicament for preventing, slowing or treating a disease or condition in a subject, said disease or condition being PAI-1. Use in relation to overexpression of
2. The use according to item 1, wherein the overexpression of PAI-1 occurs in blood or damaged tissue.
3. Use according to item 1 or 2, wherein the subject is a non-plasminogen deficient subject.
4. Use according to any one of items 1 to 3, wherein the subject has plasma plasminogen activity above 70% of normal plasminogen activity levels.
5. Item 5. The disease according to any one of Items 1 to 4, wherein the disease or the condition associated with overexpression of PAI-1 is a disease associated with a decrease in remodeling ability of blood vessels or tissues, or a metabolic disorder or a hormonal disorder. use.
6. The above-mentioned diseases associated with decreased remodeling ability of blood vessels or tissues include chronic kidney disease, glomerulosclerosis, fibrosis, tubular interstitial fibrosis, glomerulonephritis, atherosclerosis, chronic obstructive lung. Use according to item 5, which is disease (COPD), pulmonary fibrosis, chronic kidney disease (CKD), membranous nephropathy, or chronic allograft nephropathy.
7. Use according to item 5, wherein the metabolic or hormonal disorder is obesity, polycystic ovary disease, amyloidosis, Alzheimer's disease, alopecia, or aging.
8. Use according to item 5, wherein the disease is pulmonary fibrosis.
9. Use according to item 8, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.
10. Use according to any one of items 8 or 9, wherein plasminogen, a variant thereof or an analogue thereof is used in combination with an antifibrotic agent.
11. Use according to item 10, wherein the anti-fibrotic agent is pirfenidone.
12. A plasminogen, variant or analog thereof for use in the prevention, delay of progression, or treatment of a disease or condition in a subject associated with overexpression of PAI-1.
13. A method of preventing, slowing progression or treating a disease or condition in a subject, wherein said disease or condition is associated with overexpression of PAI-1 and plasminogen to the subject, a variant thereof. Alternatively, the above method comprising administration of an analog.
14. Use of plasminogen, a variant thereof or an analogue thereof for the preparation of a medicament for downregulating PAI-1 overexpression in injured tissue of a subject.
15. Plasminogen, a variant thereof or an analogue thereof for use in down-regulating PAI-1 overexpression in a damaged tissue of a subject.
16. A method of downregulating PAI-1 overexpression in injured tissue of a subject, comprising the administration of plasminogen, a variant thereof or an analogue thereof to the subject.

Further aspects of the invention will be apparent to those skilled in the art from the following detailed description, claims and generalizations herein.

Figure 3 shows plasma concentrations of endogenous mouse plasminogen in bleomycin-induced mice (Bleomycin) and non-injured mice (Control (control)). FIG. 6 shows plasma concentrations of PAI-1 in bleomycin-induced mice (Bleo) and non-injured mice (Control). FIG. 3 shows the concentration of PAI-1 in bronchoalveolar lavage fluid (BALF) of bleomycin-induced mice (Bleo) and non-injured mice (Control). Inflammation of (i) normal mice (Control), (ii) bleomycin-induced mice (Bleo), and (iii) bleomycin-induced mice (Bleo+Plg) treated with 60 mg/kg plasminogen on days 10 and 14. The collagen mRNA expression in sexual lesions is measured and shown 21 days after bleomycin injury. (I) normal mice (Control), (ii) bleomycin-induced mice (Bleo), and (iii) bleomycin-induced mice treated with 6, 20 or 60 mg/kg plasminogen on days 10 and 14 (Bleo+Plg). ), CTGF mRNA expression in inflammatory lesions was measured 21 days after bleomycin injury and shown. (I) normal mice (Control), (ii) bleomycin-induced mice (Bleo), and (iii) bleomycin-induced mice treated with plasminogen at 6, 20 or 60 mg/kg on days 10 and 14 ( (Bleo+Plg), IL-6 mRNA expression in inflammatory lesions is shown measured 21 days after bleomycin injury. (I) normal mice (Control), (ii) bleomycin-induced mice (Bleo), and (iii) bleomycin-induced mice treated with plasminogen at 6, 20 or 60 mg/kg on days 10 and 14 ( (Bleo+Plg), PAI-1 mRNA expression in inflammatory lesions, measured 21 days after bleomycin injury, is shown. (I) normal mouse (Control), (ii) bleomycin-induced mouse (Bleo), (iii) bleomycin-induced mouse (Bleo-Plasminogen) treated with 6 mg/kg of plasminogen, and (iv) 400 mg/kg of pirfenidone. PAI-in inflammatory lesions of bleomycin-induced mice treated with (Bleo-Pirfenidone) and (v) bleomycin-induced mice treated with a combination of 6 mg/kg plasminogen and 400 mg/kg pirfenidone (Bleo-Plasminogen+Pirfenidone). 1 mRNA expression is measured and shown 28 days after bleomycin injury. (I) normal mouse (Control), (ii) bleomycin-induced mouse (Bleo), (iii) bleomycin-induced mouse (Bleo-Plasminogen) treated with 6 mg/kg plasminogen, (iv) 400 mg/kg of pirfenidone IL-6 in inflammatory lesions of treated bleomycin-induced mice (Bleo-Pirfenidone), (v) bleomycin-induced mice (Bleo-Plasminogen+Pirfenidone) treated with a combination of 6 mg/kg plasminogen and 400 mg/kg pirfenidone. mRNA expression is measured and shown 28 days after bleomycin injury.

The present disclosure includes plasminogen, variants thereof, or analogs thereof for downregulating PAI-1 overexpression in tissues or plasma, and preferably when PAI-1 overexpression is associated with a disease or condition. The use of the drug is disclosed. The present disclosure discloses the use of plasminogen, variants thereof, or analogs thereof for the prevention, delay of progression or treatment of diseases or conditions associated with PAI-1 overexpression in tissues or plasma.

The sources of plasminogen are diverse and include, but are not limited to, purification from blood, plasma or fractions thereof; synthetic production; or recombinant preparation. Variants include, but are not limited to, amino acid substitution(s), truncations or extensions. Plasminogens include Glu plasminogen and Lys plasminogen. Plasminogen may also be found in its full length form (1-791) called Glu-plasminogen; or if found in a shorter version called Lys-plasminogen (78-791). There is also. Variants of plasminogen and analogs of plasminogen designated by the present invention include those having plasminogen activity.

PAI-1 overexpression is synonymous with increased PAI-1 levels or above normal PAI-1 levels or overexpressed PAI-1 levels. Said PAI-1 level refers to the PAI-1 level found in either plasma or tissue of the subject, preferably injured tissue. The reference range for PAI-1 in blood is 2-15 AU/mL. Increased PAI-1 activity is observed in the elderly. Normal plasma concentrations are 5-40 ng/mL, and mean PAI-1 levels of 50-60 ng/mL are not uncommon in middle-aged male subjects. There is a well-known circadian variability in plasma PAI-1 and this variability in PAI-1 activity is responsible for the diurnal variability in net fibrinolytic activity. PAI-1 levels peak early in the morning and correspond to the nadir of net fibrinolytic activity, while the afternoon drop in plasma PAI-1 corresponds to the peak of endogenous fibrinolysis. (Vaughan 2005, PAI-1 and atherothrombosis. J. of Thrombosis and Haemostasis, 3: 1879-1883). In one embodiment, blood PAI-1 overexpression (or PAI-1 overexpression level) is at least 25% above a known normal level for the type of subject (elderly subject, middle-aged male subject, etc.), Corresponds to 50%, 100% or 200% higher levels. In one embodiment, blood PAI-1 overexpression corresponds to a level of PAI-1 of greater than 50 ng/mL blood. In one embodiment, overexpression of PAI-1 in blood corresponds to a level of PAI-1 of greater than 70 ng/1 mL/mL blood. In one embodiment, blood PAI-1 overexpression corresponds to a level of PAI-1 of greater than 100 ng/mL of blood. In one embodiment, blood PAI-1 overexpression corresponds to a level of PAI-1 of greater than 150 ng/mL blood. In one embodiment, overexpression of PAI-1 in blood corresponds to a level of PAI-1 of greater than 200 ng/mL of blood. In subjects suffering from arteriosclerosis, tissue PAI-1 levels per 100 mg tissue were detected at 99±58 ng in 11 atherosclerotic aortas and 38±20 ng in 5 normal aortas (p< 0.05) (Shireman et al. 1996, Elevated levels of plasmaminogen-activator inhibitor type 1 in atherosclerotic aorta. J. of Vasc. Surg., 8:5, 8:10). In one embodiment, tissue PAI-1 overexpression corresponds to a level of greater than 50 ng PAI-1 per 100 mg tissue. In one embodiment, overexpression of tissue PAI-1 corresponds to a level of PAI-1 of greater than 70 ng/100 mg tissue. In one embodiment, tissue PAI-1 overexpression corresponds to a level of PAI-1 of greater than 100 ng per 100 mg of tissue. In one embodiment, tissue PAI-1 overexpression corresponds to a level of greater than 150 ng PAI-1 per 100 mg tissue. In one embodiment, tissue PAI-1 overexpression corresponds to a level of PAI-1 of greater than 200 ng per 100 mg of tissue.

In one embodiment, the present invention provides plasminogen deficiency; or low plasma plasminogen activity levels; or plasma levels below plasma plasminogen activity in non-plasminogen deficient normal subjects. Exclude prophylaxis or treatment of subjects with plasminogen activity levels (termed "normal plasminogen activity"). In other words, the invention relates to the prevention, delay of progression, or treatment of a subject with non-plasminogen deficiency. Non-plasminogen deficient subjects can be designated as subjects with normal plasma plasminogen levels, or as normal subjects. Due to variability in plasminogen activity in normal subjects, normal plasminogen activity is preferably calculated on a pool of plasma collected from normal or healthy subjects who are non-plasminogen deficient. The pool of plasma is preferably collected from a sufficient amount of subjects to normalize the variability seen in an individual. Preferably, said pool of plasma is collected from at least 240 non-plasminogen deficient normal subjects, eg 120 healthy adult men and 120 healthy adult women covering from 20 to 80 years old. It In another embodiment, normal plasminogen activity corresponds to mean or mean plasminogen activity determined in a population of healthy or non-plasminogen deficient subjects. Several methods of measuring plasminogen activity are known in the art. For example, plasminogen activity is commonly determined by chromogenic or fluorogenic assays. The pool of subjects for the determination of normal plasminogen activity, and the method of measuring plasminogen activity which is preferably used according to the present invention is described in plasminogen specific metric (enzyme; procedure) (Criteria) in human plasma. for specific measurement of plasminogen (enzymatic: procedure), Electronic Journal Of the International Federation Of Clinical Chemistry And Laboratory Medicine, Vol.12, No.3,2000:. www.ifcc.org/ifccfiles/docs/plasminogen.pdf described in In one embodiment, a low plasma plasminogen activity level is 70% or less of normal plasminogen activity, 60% or less of normal plasminogen activity, 50% or less of normal plasminogen activity, normal plus. Corresponding to 40% or less of minogen activity, 35% or less of normal plasminogen activity, or 30% or less of normal plasminogen activity. Included are subjects who have sexual plasminogen deficiency or acquired plasminogen deficiency.

In one embodiment, the invention excludes prophylaxis or treatment of a subject having a congenital plasminogen deficiency or an acquired plasminogen deficiency. In one embodiment, the invention provides wood conjunctivitis, disseminated intravascular coagulation (DIC), sepsis, leukemia, vitreous disease, liver disease, Argentine hemorrhagic fever, hyperthyroidism, post L-asparaginase therapy (post L). -Asparaginase therapy), thrombolytic events, Kawasaki disease, burns and severe burns, ectopic ossification or ossifying myositis, hyalinosis, neonatal respiratory disease syndrome (NRDS), sepsis, thrombolytic therapy, stroke, acute Lung injury (ALI), acute respiratory distress syndrome (ARDS), diabetes (types 1, 1.5, 2, and 3), fulminant liver failure, Bad Chiari syndrome, microangiogenic hemolytic anemia (MAHA), Acquired plasminogen deficiency, plasma plasminogen activity level below normal plasminogen activity level, in any subject or subject with congenital plasminogen deficiency due to atypical hemolytic uremic syndrome, or wound healing Or preclude prophylaxis or treatment of plasma plasminogen activity levels corresponding to 70% or less of normal plasminogen activity levels. In one embodiment, the subject is a non-plasminogen deficient normal subject and has plasma plasminogen activity greater than 70% of normal plasminogen activity levels.

The term “subject” includes organisms in need of treatment as disclosed herein, eg, organisms with damaged organs. The term "subject" includes animals such as mammals or birds. Preferably, the subject is a mammal, including, but not limited to, humans, horses, dogs and cats. In some embodiments, the mammal is not a mouse. More preferably, the subject is a human.

In one embodiment of the invention, plasminogen is administered by one of the following routes of administration intravenous, intraperitoneal, subcutaneous, nasal, pulmonary, or rectal. In one embodiment, plasminogen is administered subcutaneously by a device adapted for sustained release, continuous, multiple or single delivery; or by injection with a syringe. In one embodiment, plasminogen is administered intravenously by a device adapted for slow infusion, bolus, single or multiple delivery.

Plasminogen may be administered once or in multiple doses. The repeated doses are preferably administered at the following frequencies: daily, every other day, twice weekly, or weekly. In order to reach the desired level quickly, it may be desirable to choose a frequent administration at the beginning of the treatment, after which the frequency of administration is reduced to the desired degree. In another embodiment, the frequency of administration may be reduced after a period of time in which some clinical benefit is achieved. Conversely, in other embodiments, the frequency of dosing may be increased after a period of time when the desired clinical benefit is rarely achieved.

In one embodiment, the therapeutically effective dose is between about 0.5-20 mg/kg; or 0.5-15 mg/kg; or 0.5-10 mg/kg; or 2-10 mg/kg. Or 3-10 mg/kg; or 3-8 mg/kg; or 4-8 mg/kg; or 5-8 mg/kg; or 6-7 mg/kg; or about 2.0 mg/kg; or about 2.5 mg/ or about 3.0 mg/kg; or about 3.5 mg/kg; or about 4.0 mg/kg; or about 4.5 mg/kg; or about 5.0 mg/kg; or about 5.5 mg/kg; Or about 6.0 mg/kg; or about 6.5 mg/kg, or about 6.6 mg/kg; or about 7.0 mg/kg; or about 7.5 mg/kg; or about 8.0 mg/kg; or about 8.5 mg/kg; or about 9.0 mg/kg; or about 9.5 mg/kg; or about 10.0 mg/kg. Multiple doses may be suitable to maintain plasma plasminogen levels for a desired period of time. Efficient plasma in reducing the observed symptoms, achieving treatment of a disorder or condition associated with PAI-1 overexpression, or preventing a condition or disorder associated with overexpression of PAI-1. It can be determined based on the accumulation of plasminogen levels. Repeated doses may be administered daily, every other day, every two days, twice weekly, weekly, or biweekly, preferably between daily and weekly. In one embodiment, the dose is administered daily, about 0.5 to about 5 mg/kg, or about 3 to about 4 mg/kg, or about 6 to about 7 mg/kg, or about 3.5 mg/kg, or about 5 mg. /Kg, or about 6 mg/kg, or about 6,6 mg/kg. In one embodiment, the dose is administered every other day and is from about 3 to about 7 mg/kg, or about 6 to about 7 mg/kg, or about 4 to about 6 mg/kg, or about 5 mg/kg, or about 6 mg/kg. kg, or about 6,6 mg/kg. In one embodiment, the dose is administered twice weekly, about 4 to about 8 mg/kg, or about 5 to about 7 mg/kg, or about 6 to about 7 mg/kg, or about 6 mg/kg, or about 6, It is 6 mg/kg. In one embodiment, the dose is administered weekly, about 5 to about 10 mg/kg, or about 6.5 to about 8.5 mg/kg, or about 6 to about 7 mg/kg, or about 6 mg/kg, or about 6. 0.6 mg/kg, or about 7.5 mg/kg.

In one embodiment, the level of peak plasminogen activity is about 5 times normal plasminogen activity (about 500%), about 3 times normal plasminogen activity (about 300%), normal plasminogen 2.5 times the activity (about 250%), about 2 times the normal plasminogen activity (about 200%), about 1.75 times the normal plasminogen activity (about 175%), the normal plasminogen About 1.5 times (about 150%) of the gen activity, about 1.25 times (about 125%) of the normal plasminogen activity, or about 1 time (about 100%) or less of the normal plasminogen activity. Is desirable.

As used herein, the term "therapeutically effective amount" is for treating, delaying or preventing a particular disorder, disease or condition associated with reduced PAI-1 levels, or for biological effects. The amount of the compound when administered to a subject to exert a sufficient effect to achieve such treatment or prevention of the disorder, disease or condition, or to exert a biological effect. Means quantity. Biological effects specified by the present invention include modulation of PAI-1 overexpression in tissues or plasma, reduction of fibrotic lesions, and/or stimulation of ECM degradation in fibrotic organs. Dosages and therapeutically effective amounts are, for example, the activity of the particular drug used, the age, weight, general health, sex, and diet of the subject, diet, time of administration, route of administration, excretion rate, and, where applicable, any Drug combination, the effect the practitioner wants the compound to have on the subject, the properties of the compound (eg, bioavailability, stability, potency, toxicity, etc.) and the particular subject the subject is suffering from. It can vary depending on various factors, including the disorder(s). Further, the therapeutically effective amount depends on the subject's blood parameters (eg, calcium level, lipid profile, insulin level, blood glucose, coagulation factors, fibrinolytic factors), severity of pathology, organ function, or underlying disease or complications. There is a case. Such suitable doses can be determined using available assays, including those described herein. When administering plasminogen to a human, the physician may, for example, prescribe a relatively low dose initially and then increase the dose until a suitable response is obtained. The dose administered will ultimately be at the discretion of the healthcare practitioner. However, it is generally envisioned that the dose of plasminogen and its frequency of administration may be within the ranges given above for therapeutically effective doses.

In one embodiment of the invention plasminogen is not radiolabeled. In one embodiment of the invention, the plasminogen is a pharmaceutical that is free or substantially free of additional proteins, ie, proteins other than plasminogen, preferably albumin-free or substantially free. Included in the composition. In one embodiment of the invention, plasminogen is included in a pharmaceutical composition that is free or substantially free of aprotinin. In one embodiment of the invention, plasminogen is included in a pharmaceutical composition that is substantially free of trypsin inhibitor. In one embodiment of the invention, plasminogen is included in a pharmaceutical composition that is free or substantially free of serine protease inhibitors. In one embodiment of the invention, plasminogen is included in a pharmaceutical composition that is free or substantially free of plasmin. In one embodiment of the invention, plasminogen is included in the pharmaceutical composition at a concentration of detergent that is free or substantially free of detergent, eg, less than 0.01 mM. The term "substantially free" shall mean that its content is below the level of detection or below the level of quantitation.

As used herein, the term “pharmaceutical composition” refers to a preparation of plasminogen ready for commercialization or administration. The pharmaceutical composition may include at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient. As used herein, the term "pharmaceutically acceptable carrier", "pharmaceutically acceptable diluent" or "pharmaceutically acceptable excipient" includes, but is not limited to. , Adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents. Agents, solvents, emulsifiers, encapsulating agents, liposomes, cyclodextrins, encapsulated polymeric delivery systems and/or polyethylene glycol matrices, which are acceptable for use in the subject and, preferably, in humans Allowed for use. The term “acceptable for use” is preferably listed in compounds approved or approvable by federal or state regulatory agencies, or in the United States Pharmacopeia or other generally accepted pharmacopoeia. Refers to a compound that exists. The pharmaceutically acceptable vehicle may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and/or vegetable oils. Good. Additional examples of pharmaceutically acceptable vehicles include, but are not limited to, USP water for injection; aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection. , Dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, polypropylene glycol; and non-aqueous vehicles such as, but not limited to, Although not, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Prevention of the action of microorganisms can be achieved by the addition of antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyhydric alcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. The nature and concentration of the pharmaceutically acceptable excipient(s) are selected to provide acceptable protein stability, ionic strength, and pH.

The term “about” as used herein shall preferably define ±5% of the relevant value, ±10% of the relevant value, or ±15% of the relevant value.

The following examples further illustrate the practice of this invention, but are not intended to limit it.

Example 1: Preparation of Plasminogen Plasminogen is purified from pooled human plasma supplied by an FDA/EMA approved US Plasma Collection Center. The purity obtained is over 95%. Purified plasminogen is composed of Glu-plasminogen, where 95% or more of plasminogen is in the monomeric form. Glu-plasminogen was prepared as previously described in PCT Publication WO 2006/120423.

Human plasminogen is composed of approximately 75 milligrams per vial of lyophilized plasminogen. Prior to infusion, the lyophilized powder in vials is reconstituted with 12.5 mL water for intravenous injection. The final composition contains sodium citrate, sodium chloride, glycine, and sucrose to provide acceptable protein stability, ionic strength, and pH.

Example 2: Effect of plasminogen in a mouse model of pulmonary fibrosis Pulmonary fibrosis in mice is induced by intratracheal instillation of bleomycin in mice. Bleomycin is a well-known anticancer drug. First, bleomycin injury causes acute inflammation immediately on day one. Then, by about day 10 to day 14, a fibrotic event occurs, causing a chronic fibrotic condition. Bleomycin-induced mice are recognized as an animal model for pulmonary fibrosis. Assessment of plasminogen activity is performed by treatment of mice with plasminogen at the appropriate time points. In this study, plasminogen was administered on days 10 and 14.

Male C57BL6 mice are given an intratracheal instillation of bleomycin sulfate (0.007 mg/mouse) on day 0. On days 10 and 14, human plasminogen was administered subcutaneously at a dose of either 6 mg/kg, 20 mg/kg or 60 mg/kg. Animals were sacrificed on day 21. Collagen was quantified by histological analysis using stained Red Cyrus stain (fluorescence) and analyzed at 400X magnification. Collagen quantification was performed using 20 field samples with inflammatory lesions in the lung, segmented using Image Pro Premier software. The amount of collagen on the entire surface of the area containing inflammatory lesions has been reported. This quantitative method makes it possible to exclude the endogenous component of collagen normally contained in the tissue. Since control mice do not have inflammatory lesions, the percentage of collagen contained in inflammatory lesions is necessarily zero. Expression of CTGF, IL-6, and PAI-1 mRNA was quantified by real-time PCR using the mouse Taqman® Gene-Expression assay (Student's t-test) normalized to HPRT1 endogenous control. ..

Bleomycin has been observed to have no significant effect on plasma levels of endogenous mouse plasminogen (Fig. 1) and PAI-1 (Fig. 2). However, bleomycin significantly increased the concentration of PAI-1 in bronchoalveolar lavage fluid (BALF) (FIG. 3), which corresponds to the location of damaged tissue predicted by bleomycin injury.

Collagen expression in inflammatory lesions was measured in bleomycin-induced mice treated with 60 mg/kg plasminogen and compared to bleomycin-induced mice (Bleo) and normal mice (Control). Normal mice (Control) had no inflammatory lesions in the lungs, so zero percent collagen is reported in FIG. Bleomycin-induced mice (Bleo) showed inflammatory lesions with elevated collagen expression on day 21. This collagen expression is characteristic of the presence of fibrotic lesions in the lung. The collagen expression level in inflammatory lesions was significantly reduced by administering 60 mg/kg of plasminogen on days 10 and 14 of bleomycin-induced mice (Bleo+Plg).

The mRNA expression of CTGF, which is a fibrosis promoting marker, was measured and reported in FIG. It has been shown that CTGF mRNA expression in bleomycin-induced mice (Bleo) is increased compared to CTGF levels in normal mice (Control). Administration of plasminogen at either 6, 20, or 60 mg/kg significantly reduced CTGF overexpression in bleomycin-induced mice (Bleo+Plg) to levels comparable to CTGF levels in normal mice (Control). did. This response is not dose-dependent and variability can occur due to the small number of study animals.

MRNA expression of IL-6, a marker of pro-fibrotic and pro-inflammatory, was measured and reported in FIG. IL-6 has been shown to be highly overexpressed in bleomycin-induced mice (Bleo) compared to IL-6 levels in normal mice (Control). Administration of plasminogen at either 6, 20, or 60 mg/kg significantly reduced IL-6 mRNA expression in bleomycin-induced mice treated with plasminogen (Bleo+Plg). This response is not dose-dependent and variability can occur due to the small number of study animals.

The effect of plasminogen on PAI-1 mRNA expression is reported in FIG. In the present study, it was reconfirmed that PAI-1 expression was increased in the injured tissue (lung) of bleomycin-induced mice (Bleo) compared to the lungs of uninjured normal mice (Control). This result is consistent with the PAI-1 levels observed in BALF of bleomycin-induced mice (Bleo) reported in FIG. PAI-1 overexpression in bleomycin-induced mice is reduced by administration of all doses of plasminogen (Bleo+Plg). The lack of significant difference between doses suggests that plasminogen has already reached its maximal effect at low doses (6 mg/mkg). These results indicate that plasminogen downregulates PAI-1 overexpression without inhibition.

These results indicate that plasminogen directly affects the reduction of pulmonary fibrosis in a bleomycin-induced pulmonary fibrosis model, including idiopathic pulmonary fibrosis (IPF), and any disease associated with overexpression of PAI-1. It is shown that it may offer potential as a new therapeutic method for the prevention, delay of progression and treatment of lung fibrosis. These data also suggest that plasminogen is effective in regulating PAI-1 in diseases associated with increased PAI-1.

Example 3: Effect of plasminogen in combination with a fibrotic agent in a mouse model of pulmonary fibrosis Pulmonary fibrosis in mice is induced by intratracheal instillation of the anti-cancer agent bleomycin in mice. This experiment was performed as described in Example 2, except that mouse plasminogen was administered. Male C57BL6 mice are given an intratracheal instillation of bleomycin sulfate (0.007 mg/mouse) on day 0. Mouse plasminogen was administered subcutaneously on days 7, 10, 13, 16, 19, 23 at a dose of 6 mg/kg alone or in combination with oral administration of 400 mg/kg of pirfenidone (once daily, 7th to 27th day). Animals were sacrificed on day 28. Collagen was quantified by histological analysis using a stained Red Cyrus stain (fluorescence) and analyzed as described in Example 2. IL-6 mRNA expression was quantified by real-time PCR using the mouse Taqman® Gene-Expression assay, normalized to HPRT1 endogenous control (Student's t-test).

In this experiment, the combination of plasminogen and pirfenidone (antifibrotic compound) was tested. The results are reported in FIG. 8 and show that both compounds are effective in reducing fibrosis when administered alone in a bleomycin-induced lung fibrosis mouse model (Bleo+Plasminogen and Bleo+Pirfenidone). It can be noted that. Furthermore, FIG. 8 shows that the combination of plasminogen and pirfenidone (Bleo+Plasminogen+Pirfenidone) has an additive effect on the reduction of collagen expression in inflammatory lesions of the lung.

According to FIG. 9, it was reported that plasminogen inhibits IL-6 mRNA expression (Bleo+Plasminogen). The effect of pirfenidone on IL-6 expression (Bleo+Pirfenidone) was not significantly different from IL-6 levels in naive bleomycin-induced mice (Bleo). However, FIG. 9 demonstrated that the combination of plasminogen and pirfenidone (Bleo+Plasminogen+Pirfenidone) had an additive anti-fibrotic effect, as shown by a significant increase inhibition of IL-6 expression. IL-6 levels in bleomycin-induced mice treated with the combination of plasminogen and pirfenidone (Bleo+Plasminogen+Pirfenidone) were reduced to levels comparable to those of normal mice (Control).
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Although headings are included herein to assist with positioning with reference to particular sections, these headings are not intended to limit the scope of the concepts described herein. , These concepts may be applicable to other sections throughout the specification. Therefore, the present invention is not intended to be limited to the embodiments set forth herein, but should be given the broadest scope consistent with the principles and novel features disclosed herein.

The singular forms “a, an: indefinite article” and “this, the: definite article” include the corresponding plural references unless the context clearly dictates otherwise. ..

Unless otherwise indicated, all numbers used in the specification and claims to describe amounts of components, reaction conditions, concentrations, properties, etc. are understood to be modified in all cases by the term "about". It should be. At a minimum, the digit numbers reported should be taken into account and ordinary rounding techniques applied to at least interpret each numerical parameter. Therefore, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending on the properties sought. Although the numerical ranges and parameters indicating the wide range of embodiments are approximations, the numerical values shown in the specific examples are reported as accurately as possible. However, numerical values inherently contain certain errors due to variations in experiments, test measurements, statistical analysis, and the like.

It should be understood that the examples and embodiments described herein are for purposes of illustration only, and various modifications or alterations taking into account thereof will be suggested to those skilled in the art, the invention and the appended claims. It is understood to be included in.

Claims (16)

Use of plasminogen, a variant or analogue thereof for the preparation of a medicament for preventing, slowing or treating a disease or condition in a subject, said disease or condition being PAI-1. Use in relation to overexpression of The use according to claim 1, wherein the overexpression of PAI-1 occurs in blood or injured tissue of a subject. Use according to claim 1 or 2, wherein the subject is a non-plasminogen deficient subject. The use according to claim 3, wherein the subject has plasma plasminogen activity greater than 70% of normal plasminogen activity levels. 5. The disease or condition associated with overexpression of PAI-1 is a disease associated with reduced remodeling capacity of blood vessels or tissues, or a metabolic or hormonal disorder. Use of. The above-mentioned diseases associated with decreased remodeling ability of blood vessels or tissues include chronic renal disease, glomerulosclerosis, fibrosis, tubular interstitial fibrosis, glomerulonephritis, atherosclerosis, chronic obstructive lung. The use according to claim 5, which is a disease (COPD), pulmonary fibrosis, chronic kidney disease (CKD), membranous nephropathy, or chronic allograft nephropathy. The use according to claim 5, wherein the metabolic or hormonal disorder is obesity, polycystic ovary disease, amyloidosis, Alzheimer's disease, alopecia, or aging. Use according to claim 6, wherein the disease is pulmonary fibrosis. Use according to claim 8, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis. Use according to any one of claims 8 or 9, wherein plasminogen, a variant thereof or an analogue thereof is used in combination with an antifibrotic agent. Use according to claim 10, wherein the antifibrotic agent is pirfenidone. Plasminogen, a variant thereof or an analogue thereof for use in the prevention, delay of progression or treatment of a disease or condition associated with overexpression of PAI-1 in a subject. A method of preventing, slowing progression or treating a disease or condition in a subject, wherein said disease or condition is associated with overexpression of PAI-1 and plasminogen to the subject, a variant thereof Or the above method comprising administration of an analogue thereof. Use of plasminogen, a variant thereof or an analogue thereof for the preparation of a medicament for downregulating PAI-1 overexpression in injured tissue of a subject. Plasminogen, a variant thereof or an analogue thereof for use in down-regulating PAI-1 overexpression in a damaged tissue of a subject. A method of down-regulating PAI-1 overexpression in injured tissue of a subject comprising administration of plasminogen, a variant thereof or an analogue thereof to the subject.

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